Team:KIT-Kyoto/Notebook/Protocol

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Revision as of 14:59, 11 October 2014

notebook

Protocol

Miniprep

Materials

Buffer 1 250μL
Buffer 2 250μL
Buffer 3 350μL
Buffer 4 500μL
Buffer 5 700μL
Distilled water 100μL
Sample

Procedure
  • Suspend the sample in buffer 1 (1.5 ml sample tube)
  • Add buffer 2 to the sample and mix it
  • Add buffer 3 to the sample and mix it
  • Centrifuge for 5 minutes at 13,000 rpm and apply the supernatants to the column
  • Add buffer 4 and centrifuge for 1 minute at 13,000rpm then throw away the filter paper
  • Add buffer 5 and centrifuge for 1 minute at 13,000rpm then throw away the filter paper
  • Centrifuge again for 1 minute at 13,000 rpm
  • Place the column on a new sample tube and add 100 microL distilled water
  • Centrifuge for 1 minute at 6,000 rpm

Recipes for Buffer
Buffer 1 (Suspension Buffer) 50 mM Tris-HCl and 10 mM EDTA, pH 8.0 (25°C) 50 μg/ml RNase A
Buffer 2 (Lysis Buffer) 0.2 M NaOH and 1% SDS
Buffer 3 (Neutralization and Binding Buffer) 4 M guanidine hydrochloride and 0.5 M potassium acetate, pH 4.2
Buffer 4 (Wash Buffer) 5 M guanidine hydrochioride and 0.5 M potassium acetate, pH 4.2
Buffer 5 (Wash Buffer) 20 mM NaCl, 2 mM Tris-HCl, pH 7.5 (25°C) [final conentrations after addition of ethanol] with 80% ethanol

PCR-1

Materials

Buffer for KOD-FX-NEO 50μL
dNTP 20μL
Primer mix 1.0μL
Template DNA 0.5μL
KOD-FX-NEO (TOYOBO) 2.0μL
H2O 26.5μL
Total 100μL

Procedure
  • Add buffer, dNTP, primer mix, Template DNA, KOD-FX-NEO and distilled water to an Eppendorf tube and mix
  • Place the PCR tubes into the PCR machine and set the program

PCR Profile for KOD-FX-NEO
Predenature Denature Annealing Extension Final Extension
98.0°C KOD-FX-NEO 98.0°C 51.0°C 68.0°C 68.0°C
2 minutes 10 seconds 30 seconds 1 minutes 2 minutes
1 cycle 30 cycles 1 cycle

PCR-2

Materials

Buffer for KOD-FX-NEO 50μL
dNTP 20μL
Primer mix 1.0μL
Template DNA 0.5μL
KOD-FX-NEO (TOYOBO) 2.0μL
H2O 26.5μL
Total 100μL

Procedure
  • Add buffer, dNTP, primer mix, Template DNA, KOD-FX-NEO and distilled water to an Eppendorf tube and mix
  • Place the PCR tubes into the PCR machine and set the program

PCR Profile for KOD-FX-NEO
Predenature Denature Annealing Extension Final Extension
98.0°C KOD-FX-NEO 98.0°C 51.0°C 68.0°C 68.0°C
2 minutes 10 seconds 30 seconds 1 minutes 2 minutes
1 cycle 30 cycles 1 cycle

AGE

Materials

Sample 5μL
Agarose gel
2X Loading Buffer Triple Dye (NIPPON GENE) 5μL
1X TAE Buffer

Procedure
  • Set an agarose gel on an electrophoresis chamber
  • Add 1X TAE buffer to the electrophoresis chamber
    Note: Do not generate bubbles under the gel
  • Add 2X loading buffer to electrophoresis samples
  • Apply samples on agarose gel wells
  • Set an appropriate voltage (100v) and run the electrophoresis
  • Stop the electrophoresis when the BPB reaches 2/3 of the gel
  • Soak the gel in EtBr (ethidium bromide) and dye it for 20 minutes
  • Place plastic cooking wrap on the trans-illuminator and irradiate UV to the gel on the wrap
  • Take photographs of the gel by using a trans-illuminator

Agarose Concentration Versus Optical Range of DNA Size
Agarose Concentration (%) DNA (kbp)
0.3 5-60
0.6 1-20
1.0 0.3-7
1.5 0.2-4
2.0 0.1-2

Ligation

Materials

DNA sample (cut out from the gel)
Distilled water 5μL
DNA ligase 5μL

Procedure
  • Add DNA sample, distilled water and DNA ligase into a micro test tube and vortex
  • Ligation for 5minutes at room temperature

Transformation (E.coli)

Materials

DNA Sample 10μL
Competent cell 50μL

Procedure
  • Thaw the competent cells on ice
  • Add 10 μL of sample into thawed competent cells
  • Cool the sample tube, which contains competent cells and DNA samples, with ice for 1 hour, then Heat shock the cells by immersion in pre-hearted water bath at 42°C for 30 seconds
  • Place the tube on ice for 2 minutes to cool it down
  • At a clean bench, add 1.0 ml of LB medium into the tube and suspend it
  • Incubate the tube at 37°C for 35 minutes
  • Harvest the cells by centrifuge
  • Spread the transformed competent cells onto the agar plate and incubate it at 37°C overnight

Pre-culture

Materials

Sample
Medium 20ml

Procedure
  • Scrape samples from the agar plate and inoculate them on medium
  • Cultivate at 37°C in a shaken culture overnight

Main Culture

Materials

LB medium with appropriate antibiotics (20 ml) 100ml
Sample (E.coli cells)

Procedure
  • Scrape samples from the agar plate and inoculate them in liquid media
  • Cultivate overnight (37˚C, 120 rpm)


Protein Extraction (E.coli)

Materials

Sample
Fast Break Cell Lysis Reagent, 10X (Promega)
50mM potassium phosphate buffer (=pH6.8)
SDS sample buffer

Procedure
  • Separate samples into two and harvest by centrifuge
  • Add potassium phosphate buffer, then mix and remove medium completely
  • Add Fast Break Cell Lysis Reagent, 10X at the ratio of Fast Break Buffer Cell Lysis Readent,10X: Samples=1:9 and extract protein for 15 minutes at room temperature

Colony Sweep

Materials

Sample
Phenol/Chloroform water-saturated solution (Phe/Chl)
Cracking solution 3% w/v SDS, 50 mM Tris-base, pH12.6

Procedure
  • Dispense cracking solution (50 μL) each into sample tubes
  • Collect the sample and suspend it into cracking solution
  • Incubate at 65°C for 10 minutes
  • Add Phe/Chl and BPB pigment and vortex
  • Centrifuge at 14,000 rpm for 5 minutes
  • Apply the samples (upper layer) to agarose gel with loading dye
  • Check the bands by agar gel electrophoresis

SDS-PAGE

Materials

Sample
Separating Gel
Stacking Gel

Procedure
  • Place stacking gel on separating gel
  • Rinse the wells of gel with distilled water
  • Load prepared samples into wells
  • Run the electrophoresis (25 mA for 75 min)
  • Check the bands with CBB stain

Separating Gel
Acylamide (%) 7.5 10 12.5 15 17.5
MiliQ H2O (ml) 3.89 3.22 2.55 1.87 1.2
Acrylamide/Bis-acrylamide (30%/0.8% w/v) 1.99 2.7 3.38 4.04 4.7
1.5M Tris-HCl(pH8.8) (ml) 2 2 2 2 2
10% (w/v)SDS (μl) 80 80 80 80 80
10% (w/v) ammonium persulfate (AP) (μl) 27 27 27 27 27
TEMED (μl) 4 4 4 4 4

Stacking Gel
MiliQ H2O (ml) 2.89
30% (w/v) acrylamide (ml) 0.79
0.5M Tris-HCl(pH6.8) (ml) 1.25
10% SDS (μl) (ml) 50
10% APS (μl) (ml) 17
TEMED (μl) (ml) 5

Western Blotting

Materials

BufferⅠ
BufferⅡ
BufferⅢ
Distilled water 2ml
PBS
PBS-S
PBS-T
PBS-TS
PonceauS
PVDF membrane 1 sheet
Whatman paper 6 sheets
Hybridization bag 1
Peroxidase Stain Kit one drop for each
antiglutathione S - transferase (和光純薬工業株式会社製) 1μL

Procedure
  • Cut the gel in appropriate size
  • Add BufferⅢ and gel then shake it gently
  • Soak the membrane on ethanol then soak it in BufferⅢ and percolate
  • 2, 1, 3 Whatman papers (all in the same size) on BufferⅠ, BufferⅡ, BufferⅢ respectively. wet the surface of the blotter
  • Blot at the constant current of membrane's area ×2.5 mA
  • Dye the membrane with Ponceau S for 5 minutes rinse it with distilled water and scan it
  • Shake and wash with PBS-TS (3 minutes ×3 times)
  • Put the membrane in a hybridization bag add PBS-S antiglutathione S-transferase and shake it for one hour at room temperature
  • Shake and wash with PBS-T twice (5 min/10 min)
  • Shake and wash with PBS twice (5 min/5 min)
  • Add one drip of 3 Peroxidase Stain Kits and distilled water
  • Scan it

Reagent
  • BufferⅠ: bring to 300 ml with Tris base 10.9g, MetOH60ml/H2O
  • BufferⅡ: bring to 300 ml with Tris base 0.9g, MetOH60ml/H2O
  • BufferⅢ: bring to 300 ml with Tris base 0.91g, Boric acid10.5mg, MetOH60ml/H2O
  • PBS-S: PBS with 1% SkimMilk
  • PBS-T: PBS with 0.05% Tween20
  • PBS-TS: PBS with 0.05% Tween20+1% SkimMilk


LB Broth/ LB Medium

Materials

Tryptone final concentration: 1%(w/v)
Yeast Extract final concentration: 0.5%(w/v)
NaCl final concentration: 1%(w/v)
5M NaOH

Procedure
  • Dissolve Tryptone (1.0 g), Yeast Extract (500 mg) and NaCl (1.0 g) in distilled water (90 ml)
  • Adjust pH to 7.0 by adding 20μL of 5M NaOH
  • Fill up to 100 ml with distilled water
  • Autoclave

Note
  • To make agar plate, add agar powder at the final concentration of 2 %(w/v) in Procedure 2

YPD Broth/YPD Medium

Materials
Peptone final concentration: 2%(w/v)
Yeast extract final concentration: 1%(w/v)
Glucose final concentration: 2%(w/v)

Procedure
  • Dissolve peptone (2.0 g), yeast extract (1.0 g) and glucose (2.0 g) to distilled water (90 ml)
  • Fill up to 100 ml with distilled water
  • Sterilize by autoclave

Note
  • To make agar plate, add agar powder (final concentration: 2.0%) at procedure 1.

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